Am J Physiol Heart Circ Physiol 295: H4 –H7, 2008;
doi:10.1152/ajpheart.00499.2008.
Editorial Focus
The proverbial chicken or the egg? Dissection of the role of cell-free
hemoglobin versus reactive oxygen species in sickle cell pathophysiology
Megan L. Krajewski,1 Lewis L. Hsu,2 and Mark T. Gladwin1,3
1
Pulmonary and Vascular Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, Maryland; 2Pediatric Hematology, Marian Anderson Comprehensive Sickle Cell Center, Saint Christopher’s
Hospital for Children, Philadelphia, Pennsylvania; 3Critical Care Medicine Department, Clinical Center, National Institutes
of Health, Bethesda, Maryland
Address for reprint requests and other correspondence: M. T. Gladwin,
Pulmonary and Vascular Medicine Branch, NHLBI, Critical Care Medicine
Dept., Clinical Ctr., NIH, Bldg. 10-CRC, Rm. 5-5140, 10 Center Dr., MSC
1454, Bethesda, MD 20892-1454 (e-mail: [email protected]).
H4
superoxide is preferentially formed over NO, amplifying the
conditions of oxidant stress (33).
In their study, Kaul and colleagues (16) examine the mechanism of sickle cell vasculopathy in a transgenic mouse model
of severe SCD and find robust correlations between in vivo NO
resistance, measured by vasodilatory response to topical
application of the NO donor sodium nitroprusside (SNP),
hemolytic rate, and ROS generation. They present further
evidence that arginine supplementation improves vascular
function by ameliorating hemolysis, oxidant stress, and the NO
resistance state.
The assessment of vascular reactivity in the sickle cell
transgenic mouse revealed significantly blunted responses to
both acetylcholine (ACh) and SNP, as well as blunted changes
in mean arterial pressure (MAP) in response to NG-nitro-Larginine methyl ester (L-NAME), consistent with a global
impairment in the NO axis and, more specifically, a resistance
to NO vasodilatory activity (evidenced by the lack of response
to an exogenous NO donor) (13, 15). The authors confirm their
previous findings of compensatory increases in eNOS and
cyclooxygenase-2 protein expression in the sickle cell transgenic mouse, as well as increased baseline arteriolar vasodilation (15), indicating a potential compensatory upregulation of
non-NO-dependent vasodilators (prostacyclin) in response to
diminished NO bioavailability.
Interestingly, arginine treatment in the sickle cell mouse
reversed the NO resistance, such that responses to ACh and
SNP were augmented following arginine treatment, with increased MAP in response to L-NAME, indicating an increased
basal and stimulated NO bioavailability.
This study demonstrates striking correlations between hemolytic rate and markers of oxidant stress in the transgenic
sickle cell mouse, including novel findings of tight associations
between plasma hemoglobin and both tyrosine nitration and
blunted SNP responsiveness. Arginine supplementation led to
improved vascular responsiveness to both endothelium-dependent and -independent vasodilation, suggesting that arginine
was directly correcting the primary NO resistance state. This
treatment effect was associated with a 50% reduction in plasma
hemoglobin and a significant decrease in tyrosine nitration,
suggesting both reduced hemolysis and oxidant stress, respectively. These findings illuminate the complex interactions of
hemolysis and oxidant stress in potentiating vascular dysfunction (Fig. 1).
The observation of an association between tyrosine nitration
and blunted vasodilatory responses to NO donors has led to the
hypothesis that superoxide formed by xanthine oxidase or
NADPH oxidase is reacting with NO to form peroxynitrite,
which in turn is nitrating tyrosine residues. Thus the nitrotyhttp://www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 18, 2017
SICKLE CELL PATHOPHYSIOLOGY comprises a complex interplay of
episodic vasoocclusive events, ischemia-reperfusion injury,
overproduction of reactive oxygen species (ROS), inflammation, endothelial activation, and hemolysis, all somehow driven
by a single amino acid substitution in the ␤-globin chain of
hemoglobin. Hemolysis and oxidative stress act synergistically
to promote vascular dysfunction in sickle cell disease (SCD).
As a result of chronic hemolysis, levels of free plasma hemoglobin are increased at baseline and nitric oxide (NO) bioavailability is diminished, producing endothelial dysfunction that
has been linked to chronic vasculopathic complications of SCD
such as pulmonary hypertension, cutaneous leg ulceration,
priapism, and sudden death (14, 18, 19, 22, 23, 25).
NO regulates vasorelaxation and also possesses antioxidant,
antiadhesive, and antithrombotic properties (33). NO is produced from the substrate L-arginine by endothelial nitric oxide
synthase (eNOS) and mediates vasorelaxation through a paracrine action on vascular smooth muscle cells underlying the
endothelium. Endothelial dysfunction, characterized by impaired vascular responsiveness resulting from decreased NO
bioavailability, is associated with atherosclerosis, diabetes mellitus, hypertension, hypercholesterolemia, smoking, and obesity, illustrating the central importance of NO in the physiological regulation of vasomotor activity (3, 6).
Unlike coronary artery disease and its risk factors, which are
associated with an impaired production of NO, SCD and other
hemolytic diseases are characterized by a primary resistance to the
action of NO (10, 13, 25, 29). The NO resistance state observed
in SCD is multifaceted, with at least two major mechanisms
contributing to impaired NO homeostasis: 1) scavenging of NO
by cell-free plasma hemoglobin, and 2) oxidant stress due to the
generation of ROS by both enzymatic and nonenzymatic pathways (11, 13, 19, 25). Hemolysis “unpackages” the red blood
cell (RBC), releasing free hemoglobin into the plasma. No
longer compartmentalized by the intact cell membrane, cellfree plasma hemoglobin rapidly reacts with and scavenges
endothelial NO. Hemolysis further impairs NO bioavailability
through the release of arginase from the RBC, which competes
with NO synthase (NOS) for the substrate arginine. Arginase I
levels and activity correlate with measures of intravascular
hemolysis in patients with SCD, and notably the lowest ratios
of arginine to ornithine are associated with pulmonary hypertension and prospective mortality (19, 20). The depletion of
arginine leads to the functional uncoupling of NOS, whereby
Editorial Focus
H5
rosines are considered footprints for superoxide formation and
superoxide-dependent NO scavenging. There is evidence suggesting that this mechanism could contribute to NO resistance
in SCD; in the sickle cell mouse, xanthine oxidase is upregulated (1) and endothelial NAPDH oxidase is implicated in
endothelial dysfunction of the cerebral microcirculation (32).
However, data from the NO biochemistry field suggests that
the major pathway to protein nitration in vivo is via hememediated peroxidase chemistry (9, 24, 31). Any heme capable
of Fenton-type chemistry can exert peroxidase chemistry,
which in the presence of nitrite will generate nitrogen dioxide
and nitrate tyrosine residues. Indeed, the myeloperoxidase
knockout mouse exhibits significant reductions in protein nitration in vivo (5, 35). We would therefore propose that the
high correlation between plasma hemoglobin and protein nitration in the current study by Kaul and colleagues (16)
indicates that plasma hemoglobin may be driving the protein
nitration rather than the superoxide-NO reaction, which forms
peroxynitrite.
We would further argue that the role of hemolysis and
plasma hemoglobin in fueling oxidant stress is underappreciated, creating a “chicken or the egg” dilemma as to whether
oxidant stress (which leads to RBC damage and subsequent
hemolysis) or hemolysis (which releases heme and free iron,
powerful catalysts of ROS generation) is central to sustaining
the vicious cycle of damage incited by hemoglobin S polymerization (Fig. 1). Although the association does not indicate
causality, we propose the results of the current study are more
AJP-Heart Circ Physiol • VOL
295 • JULY 2008 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 18, 2017
Fig. 1. A vicious cycle of hemolysis, nitric oxide (NO) resistance, and oxidant
stress, with interruption by arginine repletion. In sickle cell disease, hemoglobin S (HbS) leads to red blood cell (RBC) hemolysis, decreased NO bioavailability, and oxidant stress. Intravascular hemolysis releases cell-free hemoglobin into the plasma compartment, contributing directly to both impaired NO
bioavailability and oxidant stress. Hemolysis alters NO homeostasis through
scavenging of NO by cell-free hemoglobin and consumption of arginine by
arginase released from hemolyzed RBCs. Hemolysis drives oxidant stress
through free hemoglobin-mediated peroxidase, autooxidation, and Fenton
chemistries, producing nitrogen dioxide and tyrosine nitration. NO resistance
is aggravated by enzymatic (xanthine oxidase and NADPH oxidase) production of superoxide, which scavenges NO. Oxidant stress perpetuates the cycle
by rendering RBCs more susceptible to damage and hemolysis. Remarkably,
arginine supplementation appears to target this triad of pathology by increasing
NO formation, reducing hemolysis, and reducing oxidant stress. NOS, NO
synthase.
consistent with hemolysis driving ROS formation and protein
nitration. Intravascular hemolysis generates free heme and
redox active metals, which participate in peroxidase chemistry
(leading to lipid peroxidation), Fenton-type chemistry, and
autooxidation chemistry (31). Thus these free heme and redox
metals can mediate protein nitration under a greater range of
conditions than peroxynitrite. Moreover, the uptake of plasma
free heme or heme released by methemoglobin into endothelial
cells promotes cellular damage, increasing the susceptibility to
oxidant damage, and may directly activate xanthine oxidase
and NADPH oxidase (2). Further evidence lending support to
a predominant role of hemolysis is the increased heme oxygenase-1 (HO-1) expression in transgenic sickle cell mice (4),
a finding confirmed in this study. The liberation of free heme
by hemolysis induces the expression of HO-1, which scavenges the heme, thereby preventing its participation in redox
reactions (8). This compensatory response to the increased
heme burden degrades heme into iron, which is scavenged by
ferritin, and carbon monoxide and biliverdin, which exhibit
antioxidative properties of their own. Rather than serving as a
marker of non-NO vasodilatory activity, we suggest HO-1
better reflects the extent of hemolysis. The decrease in plasma
hemoglobin and the associated decrease in HO-1 expression
following treatment with arginine suggest a direct effect of
arginine on reducing the hemolytic rate.
In this model, arginine therapy reduces hemolysis and oxidant stress and normalizes the responsiveness to NO. This
observation should be further explored to better elucidate the
primary mechanism of action in targeting these tightly linked
processes. Does arginine inhibit oxidant stress through increased erythrocytic glutathione and glutamine (an intraerthyrocytic antioxidant) and NOS recoupling (by restoring arginine
availability), thereby reducing hemolysis secondary to (reduced)
free oxygen radical-induced damage (13, 21)? Or, is the primary effect of arginine the result of decreased hemolysis via
the inhibition of the endothelin-1/Gardos channel pathway,
thus decreasing free heme and iron and removing the catalyst
for lipid peroxidation, nitration, and autooxidation (27, 28)?
Will dissecting these interrelationships allow us to better design combinations of therapies that effectively disrupt the cycle
of hemolysis and oxidant injury?
Previous studies of arginine supplementation in experimental animal models and patients with SCD have produced
variable results. Increased NO bioavailability after the BERK
sickle mouse was treated with L-arginine was associated with
the reduction in lipid peroxidation and augmented antioxidant
activity (7). This treatment was also found to reduce RBC
density via a NO-dependent downregulation of the Gardos
channel (likely via an intermediate effect of endothelin receptors on RBCs) (26). Arginine treatment of patients with SCD
increases plasma NO metabolite levels and acutely reduces
pulmonary artery pressures (20). Recently, the enthusiasm for
the clinical application of arginine for patients with SCD has
been tempered by the results of a multicenter, blinded, phase II
clinical trial. Presented in abstract form, arginine supplementation
in pediatric patients with SCD at 0.05 or 0.1 g䡠kg⫺1 䡠day⫺1 failed
to raise blood arginine levels or show significant changes in
laboratory indexes of clinical benefit (30). However, the doses of
arginine were notably lower than the typical dosing in the
cardiovascular field, and the lack of measurable increase in
blood arginine levels suggests that a pharmacological dose of
Editorial Focus
H6
AJP-Heart Circ Physiol • VOL
bioavailability, and oxidant stress underlying vascular dysfunction. Short of inhibiting the hemolytic rate, the inhibition of
enzymatic generation of ROS alone may fail to effectively
disrupt this vicious cycle in patients with SCD.
REFERENCES
1. Aslan M, Ryan TM, Adler B, Townes TM, Parks DA, Thompson JA,
Tousson A, Gladwin MT, Patel RP, Tarpey MM, Batinic-Haberle I,
White CR, Freeman BA. Oxygen radical inhibition of nitric oxidedependent vascular function in sickle cell disease. Proc Natl Acad Sci USA
98: 15215–15220, 2001.
2. Balla J, Vercellotti GM, Jeney V, Yachie A, Varga Z, Jacob HS, Eaton
JW, Balla G. Heme, heme oxygenase, and ferritin: how the vascular
endothelium survives (and dies) in an iron-rich environment. Antioxid
Redox Signal 9: 2119 –2137, 2007.
3. Behrendt D, Ganz P. Endothelial function. From vascular biology to
clinical applications. Am J Cardiol 90: 40L– 48L, 2002.
4. Belcher JD, Mahaseth H, Welch TE, Otterbein LE, Hebbel RP,
Vercellotti GM. Heme oxygenase-1 is a modulator of inflammation and
vaso-occlusion in transgenic sickle mice. J Clin Invest 116: 808 – 816,
2006.
5. Brennan ML, Wu W, Fu X, Shen Z, Song W, Frost H, Vadseth C,
Narine L, Lenkiewicz E, Borchers MT, Lusis AJ, Lee JJ, Lee NA,
Abu-Soud HM, Ischiropoulos H, Hazen SL. A tale of two controversies:
defining both the role of peroxidases in nitrotyrosine formation in vivo
using eosinophil peroxidase and myeloperoxidase-deficient mice, and the
nature of peroxidase-generated reactive nitrogen species. J Biol Chem 277:
17415–17427, 2002.
6. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases:
the role of oxidant stress. Circ Res 87: 840 – 844, 2000.
7. Dasgupta T, Hebbel RP, Kaul DK. Protective effect of arginine on
oxidative stress in transgenic sickle mouse models. Free Radic Biol Med
41: 1771–1780, 2006.
8. Durante W, Kroll MH, Christodoulides N, Peyton KJ, Schafer AI.
Nitric oxide induces heme oxygenase-1 gene expression and carbon
monoxide production in vascular smooth muscle cells. Circ Res 80:
557–564, 1997.
9. Espey MG, Xavier S, Thomas DD, Miranda KM, Wink DA. Direct
real-time evaluation of nitration with green fluorescent protein in solution
and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms. Proc Natl Acad Sci USA 99: 3481–3486, 2002.
10. Gladwin MT. Deconstructing endothelial dysfunction: soluble guanylyl
cyclase oxidation and the NO resistance syndrome. J Clin Invest 116:
2330 –2332, 2006.
11. Gladwin MT, Schechter AN, Ognibene FP, Coles WA, Reiter CD,
Schenke WH, Csako G, Waclawiw MA, Panza JA, Cannon RO 3rd.
Divergent nitric oxide bioavailability in men and women with sickle cell
disease. Circulation 107: 271–278, 2003.
12. Gramaglia I, Sobolewski P, Meays D, Contreras R, Nolan JP, Frangos
JA, Intaglietta M, van der Heyde HC. Low nitric oxide bioavailability
contributes to the genesis of experimental cerebral malaria. Nat Med 12:
1417–1422, 2006.
13. Hsu LL, Champion HC, Campbell-Lee SA, Bivalacqua TJ, Manci EA,
Diwan BA, Schimel DM, Cochard AE, Wang X, Schechter AN,
Noguchi CT, Gladwin MT. Hemolysis in sickle cell mice causes pulmonary hypertension due to global impairment in nitric oxide bioavailability.
Blood 109: 3088 –3098, 2007.
14. Kato GJ, Gladwin MT, Steinberg MH. Deconstructing sickle cell
disease: reappraisal of the role of hemolysis in the development of clinical
subphenotypes. Blood Rev 21: 37– 47, 2007.
15. Kaul DK, Liu XD, Chang HY, Nagel RL, Fabry ME. Effect of fetal
hemoglobin on microvascular regulation in sickle transgenic-knockout
mice. J Clin Invest 114: 1136 –1145, 2004.
16. Kaul DK, Zhang X, Dasgupta T, Fabry ME. Arginine therapy of transgenic-knockout sickle mice improves microvascular function by reducing
non-nitric oxide vasodilators, hemolysis, and oxidative stress. Am J Physiol
Heart Circ Physiol (May 2, 2008). doi:10.1152/ajpheart.00162.2008.
17. Lin EE, Rodgers GP, Gladwin MT. Hemolytic anemia-associated pulmonary hypertension in sickle cell disease. Curr Hematol Rep 4: 117–125,
2005.
18. Machado RF, Kyle Mack A, Martyr S, Barnett C, Macarthur P,
Sachdev V, Ernst I, Hunter LA, Coles WA, Nichols JP, Kato GJ,
Gladwin MT. Severity of pulmonary hypertension during vaso-occlusive
295 • JULY 2008 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 18, 2017
arginine was not achieved. An assessment of efficacy cannot be
made with a homeopathic dosing regimen. Questions remain as
to whether a higher dose of arginine might have been more
effective or whether a lack of effect is due to a lesser degree of
arginine deficiency in a pediatric population. Based on the
findings of this study by Kaul and colleagues (16), we would
propose targeting arginine therapy to patients with SCD with
hyperhemolysis and elevated endothelin-1 levels, as these
patients may potentially derive the most therapeutic benefit.
Further insight into the cause-and-effect relationship between hemolytic rate and oxidant stress is applicable to our
understanding of the mechanisms driving other hemolytic
disease states. The data suggest a role for hemolysis in the
pathogenesis of endothelial dysfunction in many of these
diseases; for example, pulmonary hypertension is a common
complication of several chronic and hereditary hemoglobinopathies, including SCD, thalassemia major and intermedia, paroxysmal nocturnal hemoglobinuria, hereditary spherocytosis,
and microangiopathic hemolytic anemia (17). Low NO bioavailability has been implicated in the development of experimental cerebral malaria (12). In addition to decreased NO
bioavailability, malaria and SCD share several features, including arginine depletion, endothelial dysfunction, increased expression of adhesion molecules, and microvascular occlusion,
suggesting the possibility of a common mechanism of disease.
A clinical study reported last year demonstrated the improved
reactive hyperemia responses in individuals with moderately
severe malaria and endothelial dysfunction following treatment
with parenteral arginine, illustrating another example of a
potential role for decreased arginine in impaired NO bioavailability in human disease (34).
As this article highlights, hemolysis and oxidative stress are
intricately coupled in promoting vascular dysfunction, leaving
the uncertainty about the primary mechanism (i.e., the chicken
or the egg). These relationships and the ameliorating role of
arginine are highlighted in Fig. 1. Several potential approaches
to dissect and establish the relative contributions of hemolysis
versus oxidant stress to endothelial dysfunction can be proposed. Murine models deficient in components of antioxidant
systems could be transplanted with bone marrow from transgenic sickle cell mice, assessing for relationships between
vasoreactivity, plasma hemoglobin, and protein nitration, as
was performed in Fig. 6 of the study (16). Additionally, mice
could be supplemented with an excess of antioxidants and
hemolysis induced at different rates, by different mechanisms
(e.g., alloimmune, RBC membrane defect, sickle, or thalassemia). A reductionist approach might be to compare the effects
of injecting the components released by hemolysis (free hemoglobin and RBC membrane fragments) at graded levels and
compare them in mice with different levels of oxidant stress
and antioxidant loading. Finally, in a spectrum of mice with
different levels of oxidant stress, there can be a focus on acute
versus chronic effects of hemolysis, acknowledging that HO-1
upregulation and other compensatory mechanisms can blunt
some of the hemolytic effects.
In summary, Kaul and colleagues (16) verify the strong
association of NO resistance in SCD with free hemoglobin and
further our understanding by establishing an association of NO
resistance with oxidant stress. This connection should challenge us to consider hemolysis as the driving force sustaining
the polymerization-induced cycle of hemolysis, decreased NO
Editorial Focus
H7
19.
20.
21.
22.
24.
25.
26.
27.
AJP-Heart Circ Physiol • VOL
28. Romero JR, Suzuka SM, Nagel RL, Fabry ME. Arginine supplementation of sickle transgenic mice reduces red cell density and Gardos
channel activity. Blood 99: 1103–1108, 2002.
29. Rother RP, Bell L, Hillmen P, Gladwin MT. The clinical sequelae of
intravascular hemolysis and extracellular plasma hemoglobin: a novel
mechanism of human disease. JAMA 293: 1653–1662, 2005.
30. Styles L, Kuypers F, Kesler K, Reiss U, Lebeau P, Nagel R, Fabry M.
Arginine therapy does not benefit children with sickle cell anemia—results
of the CSCC Clinical Trial Consortium Multi-Institutional Study (Abstract). Blood 110: 2252, 2007.
31. Thomas DD, Espey MG, Vitek MP, Miranda KM, Wink DA. Protein
nitration is mediated by heme and free metals through Fenton-type
chemistry: an alternative to the NO/O⫺
2 reaction. Proc Natl Acad Sci USA
99: 12691–12696, 2002.
32. Wood KC, Hebbel RP, Granger DN. Endothelial cell NADPH oxidase
mediates the cerebral microvascular dysfunction in sickle cell transgenic
mice. FASEB J 19: 989 –991, 2005.
33. Wood KC, Hsu LL, Gladwin MT. Sickle cell disease vasculopathy: a
state of nitric oxide resistance. Free Radic Biol Med 44: 1506 –1528,
2008.
34. Yeo TW, Lampah DA, Gitawati R, Tjitra E, Kenangalem E, McNeil
YR, Darcy CJ, Granger DL, Weinberg JB, Lopansri BK, Price RN,
Duffull SB, Celermajer DS, Anstey NM. Impaired nitric oxide bioavailability and L-arginine reversible endothelial dysfunction in adults with
falciparum malaria. J Exp Med 204: 2693–2704, 2007.
35. Zhang R, Brennan ML, Shen Z, MacPherson JC, Schmitt D, Molenda
CE, Hazen SL. Myeloperoxidase functions as a major enzymatic catalyst
for initiation of lipid peroxidation at sites of inflammation. J Biol Chem
277: 46116 – 46122, 2002.
295 • JULY 2008 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 18, 2017
23.
pain crisis and exercise in patients with sickle cell disease. Br J Haematol
136: 319 –325, 2007.
Morris CR, Kato GJ, Poljakovic M, Wang X, Blackwelder WC,
Sachdev V, Hazen SL, Vichinsky EP, Morris SM Jr, Gladwin MT.
Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension, and mortality in sickle cell disease. JAMA 294: 81–90, 2005.
Morris CR, Morris SM Jr, Hagar W, Van Warmerdam J, Claster S,
Kepka-Lenhart D, Machado L, Kuypers FA, Vichinsky EP. Arginine
therapy: a new treatment for pulmonary hypertension in sickle cell
disease? Am J Respir Crit Care Med 168: 63– 69, 2003.
Morris CR, Suh JH, Hagar W, Larkin S, Bland DA, Steinberg MH,
Vichinsky EP, Shigenaga M, Ames B, Kuypers FA, Klings ES. Erythrocyte glutamine depletion, altered redox environment, and pulmonary
hypertension in sickle cell disease. Blood 111: 402– 410, 2008.
Nolan VG, Baldwin C, Ma Q, Wyszynski DF, Amirault Y, Farrell JJ,
Bisbee A, Embury SH, Farrer LA, Steinberg MH. Association of single
nucleotide polymorphisms in klotho with priapism in sickle cell anaemia.
Br J Haematol 128: 266 –272, 2005.
Nolan VG, Wyszynski DF, Farrer LA, Steinberg MH. Hemolysisassociated priapism in sickle cell disease. Blood 106: 3264 –3267, 2005.
Pfeiffer S, Mayer B. Lack of tyrosine nitration by peroxynitrite generated
at physiological pH. J Biol Chem 273: 27280 –27285, 1998.
Reiter CD, Wang X, Tanus-Santos JE, Hogg N, Cannon RO 3rd,
Schechter AN, Gladwin MT. Cell-free hemoglobin limits nitric oxide
bioavailability in sickle-cell disease. Nat Med 8: 1383–1389, 2002.
Rivera A, Jarolim P, Brugnara C. Modulation of Gardos channel
activity by cytokines in sickle erythrocytes. Blood 99: 357– 603, 2002.
Romero JR, Rivera A, Muñiz A, Nagel R, Fabry ME. Arginine diet for
sickle transgenic mice reduces endothelin-1-stimulated Gardos channel
activity (Abstract). Blood 110: 3399, 2007.